Endochondral ossification is essential for normal skeletal development and is involved in fracture repair. On the other hand, pathological calcification occurs in osteoarthritis, atherosclerosis, and chondrocalcinosis. In order to design effective therapies for treatment of dysfunctional calcification the mechanisms involved in mineral deposition must be understood. Since matrix vesicles (MV) are intimately associated with mineral formation in many vertebrate calcifying tissues, our goal is to characterize the essential components involved in MV calcification. Our goal is to elucidate how key proteins, lipids, and electrolytes interact to form MV with the ability to induce calcium phosphate mineral formation. The first aim is to determine the 3-D structures and the thermodynamics of binding of MV annexin V with its major functional modifiers: Ca2+, Zn2+, ATP, GTP, and the phospholipids. X-ray crystallography will be used to elucidate structure-function relationships between annexin V and these important modulators of its activity and to determine the phospholipid bind site(s) in the protein. The packing arrangement of phosphatidylserine (PS): calcium (Ca):inorganic phosphate (Pi): annexin V complexes will be observed by transmission electron microscopy (TEM). Isothermal titration calorimetry will be used to obtain stoichiometric and thermodynamic values for annexin V binding to its ligands. In the second aim, refinement in the synthesis and physicochemical characterization and the molecular structure determination of the complex of PS:Ca:Pi and annexin V that constitutes the nucleational core of MV will continue. Functional MV-like structures will be synthesized by encapsulating annexin V and electrolytes into large unilamellar vesicles containing lipid profiles similar to those of native MV. Analytical techniques to be used include Fourier transform infrared spectroscopy, high resolution X-ray diffraction, TEM with EDAX analysis for Ca:P stoichiometry, and solid-state 31P-NMR to provide details of the early mineral phases induced by MV and the nucleational complex. While most of the proposed work will take place using the facilities at the University of South Carolina, we also have access to state-of-the-art X-ray and 31P-NMR facilities at the Advanced Photon Source synchrotron at Argonne National Laboratory and the high-field NMR at the Battelle Pacific Northwest National Laboratory. The objective of the proposed research is to further characterize key components and events, in many cases at the atomic level, which are critical to the mechanism of MV function. Our long-term goal is to produce synthetic MV and/or nucleational materials that can induce mineralization and promote healing of bone fractures or other recalcitrant bone injuries.